Endophytic microorganisms occur within living plant tissues without causing apparent damage to the host (Petrini, 1991). To date, endophytic yeasts have been isolated from a variety of plants, including roots of Zea mays L. (maize) (Nassar et al., 2005) and roots of Musa acuminate L. (banana) (Cao et al., 2002), and leaves of Oryza sativa L. (rice) (Tian et al., 2004), Solanum lycopersicum L. (tomato) (Larran et al., 2001), and Triticum aestivum L. (wheat) (Larran et al., 2002). In a series of studies on endophytic microorganisms in wild and hybrid Populus species, (Doty et al., 2005; Doty et al., 2009) three yeast strains were isolated.
Identification of yeast species from morphological and physiological characteristics has been complemented with and improved by molecular methods in the last 20 years. Analyses of small subunit (18S) ribosomal RNA (rRNA) gene sequences, extremely important in phylogenetic analyses of species in bacteria, generally are not adequate to differentiate yeast species (James et al., 1996; Kurtzman and Robnett, 2003). Sequencing domains 1 and 2 (D1/D2) of large subunit (26S)rRNA gene have been used by many researchers to determine yeast species because this approach is rapid and effective, and a large number of sequences are available for comparison in online databases (Kurtzman and Robnett, 1998; Fell et al., 2000; Kurtzman, 2006). The internal transcribed spacer (ITS) regions ITS1 and ITS2, flanking the 5.8S gene of rRNA, are also highly substituted and are used for yeast identification. Scorzetti et al. (2002) found that analyzing ITS sequences allowed them to detect species among basidiomycetous species more effectively than using D1/D2. For example, Sporobolomyces holsaticus Windisch ex Yarrow & Fell and Sporidiobolus johnsonii Nyland are identical in D1/D2 sequences, register 93% DNA hybridization (Boekhout, 1991), and differ in five base positions in the ITS sequences. In contrast, Rhodotorula glutinis (Fresen.) F. C. Harrison and Rhodotorula graminis Di Menna are identical in the ITS region but differ in one base position in D1/D2; they are considered to be separate species based on 35-40% DNA hybridization (Gadanho and Sampaio, 2002). Consequently, it appears useful to sequence both D1/D2 and ITS regions when distinguishing closely related species, while defining species taxonomically requires classical phenotypic information (Scorzetti et al., 2002).
Recently, the role of endophytic microorganisms in the promotion of plant growth has received increased attention. Endophytes can promote plant growth through different mechanisms, including delivery of fixed nitrogen to host plants, production of plant growth regulators, and biological control of plant pathogens (Ryan et al., 2008). Endophytic yeast strains have been shown to be able to promote the growth of maize (Nassar et al., 2005) and Beta vulgaris L. (sugar beet) (El-Tarabily, 2004) by producing plant auxins, such as indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPYA) (Nassar et al., 2005).
Efficient industrial production of biofuels, such as bioethanol, holds promise for serving the growing energy needs of the world in the near future. Ethanolic fermentation of cellulosic and lignocellulosic biomass by microorganisms such as yeast is currently employed in the industrial production of bioethanol. However, the lack of non-pathogenic microorganisms that efficiently metabolize both five carbon (pentose) and six carbon (hexose) sugars in the presence of high levels of ethanol, limits the efficiency and therefore the economic feasibility of large-scale fermentations of certain lignocellulosic carbon sources that contain high levels of hemicellulosic biomass. As an example of this, in the absence of corn, the maize plant is comprised of about 24% Xylose and 2% arabinose, both of which are pentose sugars that are poorly utilized by the industrial yeast strains currently employed (Antoni et al., Appl Microbiol Biotechnol 77:23-35 (2007)).
Several groups have attempted to traverse this problem by genetically engineering strains of Saccharomyces cerevisiae to contain enzymes necessary for efficient metabolism of xylose. In initial attempts, various bacterial xylose isomerases were expressed in S. cerevisiae (Amore et al., Appl Microbiol Biotechnol 30:351-357 (1989); Ho et al., Biotechnol Bioeng Symp 13:245-250 (1983); Moes et al., Biotechnol Lett 18:269-274 (1996); Sarthy et al., Appl Environ Microbiol 53:1996-2000 (1987); Walfridsson, et al., Appl Environ Microbiol 62:4648-51 (1996)). However, only minimal xylose metabolism was found in these recombinant yeast at temperatures suitable for industrial application.
Other groups have tried to enhance the ethanolic fermentation of S. cerevisiae by exogenously expressing P. stipitis xylose reductase (XR) and xylose dehydrogenase (XDH) (Kötter and Ciriacy, Appl Microbiol Biotechnol 38:776-783 (1993); Tantirungkij et al., J Ferm Bioeng 75:83-88 (1993); Walfridsson et al., Appl Microbiol Biotechnol 48:218-224 (1997)). These studies also failed to yield recombinant S. cerevisiae strains that utilized xylose for high yield ethanolic fermentation.
U.S. Pat. No. 7,091,014 to Aristidou et al. describes the genetic engineering of fermenting microorganisms, including S. cerevisiae and Schizosaccharomyces pombe, to express an NAD-dependent glutamate dehydrogenase (GDH) or malic enzyme (ME). These modified yeast display modest increases in ethanol and xylitol production, but do not appear to metabolize xylose any faster than control strains lacking the GDH or ME enzymes.
U.S. Pat. No. 7,253,001 to Wahlbom et al. provides genetically engineered yeast for the ethanolic fermentation of xylose. The engineered yeast of U.S. Pat. No. 7,253,001 recombinantly express exogenous genes for xylose reductase, xylitol dehydrogenase, xylulokinase, phosphoacetyltransferase, aldehyde dehydrogenase, and optionally phosphoketolase.
Similarly, U.S. Pat. No. 7,226,735 to Jeffries and Jin provides genetically engineered yeast strains comprising heterologous gene sequences encoding xylose reductase, xylitol dehydrogenase, and D-xylulokinase enzymes, which are capable of performing fermentation of xylose. U.S. Pat. No. 7,285,403 to Jeffries et al. provides similar engineered yeast strains that additionally display reduced PHO13 expression.
One drawback to using these genetically engineered yeast strains for food and beverage production is that the products, such as ethanol and xylitol, may be regulated as novel GMO (genetically modified organism) produced food. Such regulations may result in additional safety and labeling requirements that are not needed for foods produced by using unmodified organisms. As such, there remains a need in the art for methods of efficiently fermenting pentose and hexose sugars without the use of genetically modified organisms.
Xylitol, a five carbon sugar alcohol, is an increasingly utilized sugar substitute with several desirable properties. First several studies have shown that xylitol provides anticariogenic effects that promote oral health (Tanzer J M., Int Dent J. 1995 February; 45(1 Suppl 1):65-76). Secondly, xylitol metabolism is not regulated by the insulin pathway, which makes this sweetener an attractive sugar substitute for diabetics. Similarly, xylitol is an appropriate sugar substitute for individuals who suffer from glucose-6-phosphate dehydrogenase deficiencies. Finally, xylitol has fewer calories and net effective carbohydrates than does table sugar, making it a viable dietary substitute for sucrose.
Although xylitol is present in many fruits and vegetables, extraction is inefficient and uneconomical. As such, xylitol is industrially produced through the chemical reduction of xylose. Typically, xylan-containing biomass is hydrolyzed to produce a mixture of pentose and hexose sugars, including D-xylose. After enrichment, D-xylose is then converted to xylitol in a chemical process using e.g. a nickel catalyst such as Raney-nickel. Many procedures for this process have been developed, for example see U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, 4,008,285, and 3,586,537. However, the use of xylitol is still limited due to the high costs of production and purification. Accordingly, improved biotechnological processes for the production of xylitol, especially from readily available carbon sources such as corn, sugar cane, and various wood sources high in hemicellulosic biomass, are highly desirable.
Several xylose-metabolizing yeast species have been suggested for use in the production of xylitol, including species of Candida (WO 90/08193, WO 91/10740, WO 88/05467, U.S. Pat. No. 5,998,181), mutant and genetically modified Kluyveromyces (U.S. Pat. No. 6,271,007), Debaryomyces (Rivas et al., Biotechnol Bioeng. 2008 Oct. 3) and genetically modified Saccharomyces (U.S. Pat. No. 7,226,761). However, use of the above yeasts have failed to translate into economically viable industrial procedures for the biotechnological production of xylitol. As such, there remains a need in the art for processes that utilize xylose-metabolizing microorganisms in the industrial production of xylitol.
Nitrogen fixation refers to the biological process by which atmospheric nitrogen (N2) is converted into ammonia. This process is essential for life because fixed nitrogen is required for the biosynthesis of both amino acids and nucleotides and as such is required for all plant growth. Unfortunately, most plants, including industrially and commercially important crops, are unable to fix nitrogen. These plants rely on nitrogen fixation from various prokaryotes, termed diazotrophs, including species of bacteria and actinobacteria.
Due to the high fixed nitrogen requirements, fixed nitrogen is commonly a limiting resource for plant growth. To combat this, farmers typically rely on fertilizers to supplement the fixed nitrogen content of the soil used for crop growth.
Despite the need for fixed nitrogen supplementation, there are several disadvantages to the use of fertilizers and in particular chemically synthesized inorganic fertilizers. For example, synthesized nitrogen requires high levels of fossil fuels such as natural gas and coal, which are limited resources. In fact, according to the International Fertilizer Industry Association (IFA), production of synthetic ammonia currently consumes nearly 2% of the world energy production with more than 100 million metric tons of ammonia being produced in 2008.
In addition, the run-off of nitrogen-rich compounds found in fertilizers is suspected to be a major contributor to the depletion of oxygen in many parts of the ocean, especially in coastal zones, such as off the coast of the pacific northwestern region of North America. Similarly, methane and nitrous oxide emissions resulting form the use of ammonium based fertilizers may contribute to global climate change, as greenhouse gasses.
Practically speaking, the high cost of growing food crops and biomass for the production of bioenergy (i.e., bioethanol) is in part due to the high cost of fertilizers. As such, methods of nitrogen fixation and crop fertilization that reduce or eliminate the reliance on chemically synthesized fertilizers are needed to reduce the environmental, agricultural, and financial impact that accompany the use of traditional fertilizers.
The present invention provides three novel yeast isolates that are capable of metabolizing a wide range of pentose and hexose sugars, as well as novel methods for the production of bioethanol and xylitol, the fixation of nitrogen, and crop fertilization, which satisfy these and other needs in the art.